Journal: bioRxiv

Article Title: Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes

doi: 10.64898/2026.03.19.712941

Figure Lengend Snippet: A. Western blot showing knockdown of endogenous RHBDL2 protein in HaCaT cells by multiple shRNAs (01, 00, 02) compared with wild-type (WT) and vector control (V) cells. B. Statistics of the duplicate proteomics experiments. Numbers of identified and quantified proteins ranked by their topology are shown for each of the SILAC experiments, their overlap and union. Type I membrane proteins (with a signal peptide and a single transmembrane helix), which are potential rhomboid substrates, represent about 25% of the secretome in each case and are shown in pale green. Blue, type II membrane proteins; turquoise, polytopic transmembrane proteins; grey, secreted proteins; black, intracellular proteins. C. Changes in membrane protein abundance in HaCaT keratinocyte secretome induced by RHBDL2 expression. In two independent reverse experiments, WT and R2kd HaCaT cells were isotopically labelled by heavy or light lysine and arginine, the media from both populations were pooled and the lectin-enriched glycoproteins were identified and quantified by MS analysis. The abundance ratios of all transmembrane proteins identified in both experiments (i.e. the overlap of the two datasets) were plotted against each other. Two-fold enrichment was set as a significance threshold (dotted line). Membrane proteins occurring in the grey quadrant showed consistent enrichment in both experiments and represent strong candidates for RHBDL2 substrates. D. HaCaT cells were stably transfected with constructs encoding fluorescent fusions of EGFR (GFP) and RHBDL2 (mCherry) and analysed by confocal microscopy. Scale bar = 10 µm. E. Media from WT, vector control (V) and RHBDL2 knockdown (R2kd) HaCaT and MDA-MB-468 cells was concentrated and probed with an antibody raised against the EGFR ectodomain to detect RHBDL2 dependent shedding at endogenous levels of expression. F. Conditioned media from HaCaT cells were divided equally and one half was subjected to high-speed ultracentrifugation (UC) to remove membranes including exosomes. The supernatant after ultracentrifugation and the untreated medium were immunoblotted using separate primary antibodies raised against the extracellular N-terminal (NT) or the intracellular C-terminal part (CT) of EGFR. Ultracentrifugation selectively depletes the full-length form of EGFR, which is reactive against the C-terminal antibody. G . Contribution of metalloproteases to EGFR shedding. N-terminally Strep-tagged EGFR was expressed in HEK293ET cells alongside HA-tagged RHBDL2 or a catalytically inactive mutant (S187A) and cultivated for 24 hrs in the presence or absence of 10 µM BB94. H. The candidate cleavage sites were identified by mass spectrometry (MS) of the purified ectodomain ( Fig. S1 ). Candidate P1 residues were mutated to proline to produce uncleavable mutants and tested by co-overexpression with the WT or inactive mutant enzyme (S187A) in HEK293ET cells. I. An RHBDL2 dependent C-terminal EGFR fragment (open arrow) can be produced by overexpression of the wild-type enzyme in HaCaT cells, but not the S187T inactive mutant (left). RHBDL2 overexpressing cells were incubated overnight with 10 nM PR-171 (PR), 10 µM lactacystin (LC), 10 µM chloroquine (CQ) or 100 nM bafilomycin A1 (BA1) to determine the fate of the fragment (middle). The fragment can also be observed by overnight treatment with bafilomycin A1 at endogenous levels of RHBDL2 expression (right).

Article Snippet: To examine the biological relevance of this mechanism, we measured RHBDL2 activity and EGFR shedding in primary Normal Human Adult Epidermal Keratinocytes (NHEK-Ad) and hTERT-immortalized keratinocytes Ker-CT (ATCC CRL-4048), more stably accessible alternatives to primary keratinocytes.

Techniques: Western Blot, Knockdown, Plasmid Preparation, Control, Multiplex sample analysis, Membrane, Quantitative Proteomics, Expressing, Stable Transfection, Transfection, Construct, Confocal Microscopy, Mutagenesis, Mass Spectrometry, Purification, Over Expression, Produced, Incubation

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes

doi: 10.64898/2026.03.19.712941

Figure Lengend Snippet: A-C . HaCaT cell lines were seeded equally and grown to confluent monolayers over 48 h. Cells were lysed on ice in the presence of protease and phosphatase inhibitors and samples were diluted to equal concentrations of total protein. Lysates were then probed for various components of EGFR signalling pathways by western blotting. D. HaCaT cell migration was analysed by time-lapse phase-contrast microscopy over the indicated timeframe. Left panel shows the time sequence of the cell colony outline during spreading of wild-type, R2kd and vector transfected cells. Colony outlines were superimposed from first image to last image. For clarity, outlines corresponding to initial 340 min are shown in 20 min increments. Right panel shows quantification of average distance migrated after 16 h. Asterisks correspond to P values (ns=P > 0.05, *=P ≤ 0.05,**= P ≤ 0.01, ***=P ≤ 0.001 and ****= P ≤ 0.0001). E. Gap-closure assay. WT, R2kd and vector HaCaT cells were grown to confluence around removable silicone inserts. After insert removal, gap closure was observed by time-lapse microscopy over 16 h. The average distance migrated is quantified in the box and whisker plot. F. Quantification of migration of WT-HaCaT, R2kd cells and rescue cells in which shRNA refractory wild type RHBDL2 (R2kd+WT) or its inactive S187T mutant (R2kd+mut) were re-introduced into R2kd cells. RHBDL2 overexpression was induced by 5 µg/mL cumate where indicated. G. Depletion of RHBDL2 potentiates invasion of HaCaT cells into a 3D collagen matrix. Spheroids of HaCaT keratinocytes, wild-type (WT), Vector control (V), RHBDL2 knockdown (R2kd) and rescue cells (R2kd+WT, R2kd+mut) were embedded in collagen and their invasion was measured after 72 h by comparing the total area of invaded cells relative to the area of the cell spheroid at 0 h. Invasion is quantified from 5-8 spheroids in each of 3 replicate experiments (total ≥20). Statistical analyses by Tukey’s multiple comparisons test were performed using Prism software (GraphPad Software Inc.). H. HaCaT cell proliferation rate after 48 h growth in 3D collagen was assayed using the Alamar Blue assay. The level of fluorescence is proportional to the metabolic activity and hence can be used to estimate the number of cells relative to each line. The box and whisker plot is generated from 4 replicate experiments. Statistical analyses by Tukey’s multiple comparisons test were performed using Prism software (GraphPad Software Inc.).

Article Snippet: To examine the biological relevance of this mechanism, we measured RHBDL2 activity and EGFR shedding in primary Normal Human Adult Epidermal Keratinocytes (NHEK-Ad) and hTERT-immortalized keratinocytes Ker-CT (ATCC CRL-4048), more stably accessible alternatives to primary keratinocytes.

Techniques: Western Blot, Migration, Microscopy, Sequencing, Plasmid Preparation, Transfection, Time-lapse Microscopy, Whisker Assay, shRNA, Mutagenesis, Over Expression, Control, Knockdown, Software, Alamar Blue Assay, Fluorescence, Activity Assay, Generated

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes

doi: 10.64898/2026.03.19.712941

Figure Lengend Snippet: A. RHBDL2 mRNA comparison by qPCR in the Ker-CT and HaCaT cells. Gene expression was normalized to GAPDH and the level of RHBDL2 expression in HaCaT cells was used to normalize RHBDL2 expression in all other cell lines, also in panel F. The mRNA analysis was done from three biological replicates, each in three technical replicates. Error bars show standard deviation. B. and C. Immunoblotting of conditioned medium and lysate after 4 h incubation with 5 mM calcium and 1 µM ionomycin detecting shedding of EGFR in primary NHEK-Ad and immortalized keratinocytes Ker-CT that is inhibited by a RHBDL2 ketoamide inhibitor compound 11 (50 µM) confirming RHBDL2 dependency. Tubulin is was used as a loading control. D. Immunoblotting of conditioned medium after 4 h incubation of Ker-CT keratinocytes with 100 µM PLCγ activator m-3M3FBS in the absence and presence of RHBDL2 inhibitor compound 11 (50 µM). E. Immunoblotting of conditioned medium after 4 h incubation of Ker-CT with thapsigargin (2 µM), bradykinin (10 µM) or 5 mM calcium as a positive control that induces RHBDL2 dependent EGFR shedding. F. RHBDL2 mRNA detection by qPCR in the N/TERT keratinocyte derived RHBDL2 knockout (R2ko) cell lines with comparison to the HaCaT and HaCaT RHBDL2 knockdown (R2kd) cells. The mRNA analysis was done from three biological replicates, each in three technical replicates. Error bars show standard deviation. G. Immunoblotting of conditioned medium and lysate of N/TERT keratinocytes to confirm RHBDL2 dependency of EGFR shedding in these cells and to validate the RHBDL2 KO (CB and CC) generated in N/TERT keratinocytes. Constitutive (48 h) or calcium (5 mM) stimulated (4 h) shedding of EGFR is inhibited by compound 11 (50 µM) in the WT, confirming its RHBDL2 dependence.

Article Snippet: To examine the biological relevance of this mechanism, we measured RHBDL2 activity and EGFR shedding in primary Normal Human Adult Epidermal Keratinocytes (NHEK-Ad) and hTERT-immortalized keratinocytes Ker-CT (ATCC CRL-4048), more stably accessible alternatives to primary keratinocytes.

Techniques: Comparison, Gene Expression, Expressing, Standard Deviation, Western Blot, Incubation, Control, Positive Control, Derivative Assay, Knock-Out, Knockdown, Generated

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes

doi: 10.64898/2026.03.19.712941

Figure Lengend Snippet: A. Hematoxylin and eosin staining of human skin equivalent sections generated from wild type (WT) and RHBDL2 deficient (clones CB and CC) N/TERT keratinocytes. B. Quantification of qualitative assessment of morphology of human skin organotypic cultures is based on histological analysis. Detailed scoring of individual parameters in each layer of WT or RHBDL2 deficient clones was rated on a scale 1 to 18 where 18 denotes highest differentiation ( Fig. S6 ). Differentiation scores between groups were compared by one-way ANOVA; adjusted P-value: ****<0.0001, ***≤0.001, N=4 for each group.

Article Snippet: To examine the biological relevance of this mechanism, we measured RHBDL2 activity and EGFR shedding in primary Normal Human Adult Epidermal Keratinocytes (NHEK-Ad) and hTERT-immortalized keratinocytes Ker-CT (ATCC CRL-4048), more stably accessible alternatives to primary keratinocytes.

Techniques: Staining, Generated, Clone Assay

Journal: bioRxiv

Article Title: Rhomboid protease RHBDL2 is a calcium-activated suppressor of EGFR signalling in keratinocytes

doi: 10.64898/2026.03.19.712941

Figure Lengend Snippet:

Article Snippet: To examine the biological relevance of this mechanism, we measured RHBDL2 activity and EGFR shedding in primary Normal Human Adult Epidermal Keratinocytes (NHEK-Ad) and hTERT-immortalized keratinocytes Ker-CT (ATCC CRL-4048), more stably accessible alternatives to primary keratinocytes.

Techniques: Quantitative Proteomics, Membrane, Mass Spectrometry